Family 44 xyloglucanases

ABSTRACT

The present invention relates to xyloglucanases belonging to family 44 of glycosyl hydrolases and having a relative xyloglucanase activity of at least 30% between pH 5 and pH 8 are derived from the genus  Paenibacillus , especially from a strain of  Paenibacillus polymyxa  or  Paenibacillus  sp. The xyloglucanases exhibit high performance in conventional detergent compositions.

CROSS-REFERENCE TO SEQUENCE LISTING

This application contains information in the form of a sequence listing,which is submitted on a data carrier accompanying this application. Thecontents of the data carrier are fully incorporated herein by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/896,555filed Jul. 22, 2004, which is a continuation of U.S. application Ser.No. 09/784,554 filed Feb. 16, 2001: now U.S. Pat. No. 6,815,192, whichclaims priority of Danish application no. PA 2000 00291 filed Feb. 24,2000 and U.S. provisional application No. 60/185,317 filed Feb. 28,2000, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to xyloglucanases belonging to family 44of glycosyl hydrolases, preferably to enzymes exhibiting xyloglucanaseactivity as their major enzymatic activity in the neutral and alkalinepH ranges; to a method of producing such enzymes, and to methods forusing such enzymes in the textile, detergent and cellulose fiberprocessing industries.

2. Description of Related Art

Xyloglucan is a major structural polysaccharide in the primary (growing)cell wall of plants. Structurally, xyloglucans consists of acellulose-like beta-1,4-linked glucose backbone which is frequentlysubstituted with various side chains. The xyloglucans of mostdicotyledonous plants, some monocotyledons and gymnosperms are highlybranched polysaccharides in which approx. 75% of the glucose residues inthe backbone bear a glycosyl side chain at O-6. The glycosyl residuethat is directly attached to the branched glucose so residue isinvariably alpha-D-xylose. Up to 50% of the side chains in thexyloglucans contain more than one residue due to the presence ofbeta-D-galactose or alpha-L-fucose-(1-2)-beta-D-galactose moieties atO-2 of the xylose residues (C. Ohsumi and T. Hayashi, 1994, Plant andCell Physiology 35: 963-967; G. J. McDougall and S. C. Fry, 1994,Journal of Plant Physiology 143: 591-595; J. L. Acebes et al, 1993,Phytochemistry 33: 1343-1345). On acid hydrolysis, the xyloglucanextracted from cotton fibers yielded glucose, xylose, galactose andfucose in the ratio of 50:29:12:7 (Hayashi et al., 1988).

Xyloglucans produced by solanaceous plants are unusual in that typicalonly 40% of the beta-1,4-linked glucose residues bear a glycosyl sidechain at O-6. Furthermore, up to 60% of the xylose residues aresubstituted at O-2 with alpha-L-arabinose residues and some solanaceousplants, such as potato, also have xyloglucans with beta-D-galactosesubstituents at O-2 of some of the xylose residues (York et al., 1996).

Xyloglucan is believed to function in the primary wall of plants bycross linking cellulose-micro fibrils, forming a cellulose-xyloglucannetwork. This network is considered necessary for the structuralintegrity of primary cell walls (Carpita et al., 1993). Anotherimportant function of xyloglucan is to act as a repository forxyloglucan subunit oligo saccharides that are physiologically activeregulators of plant cell growth. Xyloglucan subunits may also modulatethe action of a xyloglucan endotransglycosylase (XET), a cell wallassociated enzyme that has been hypothesized to play a role in theelongation of plant cell walls. Therefore xyloglucan might play animportant role in wall loosening and consequently cell expansion (Fry etal., 1992).

The seeds of many dicotyledonous species contain xyloglucan as the majorpolysaccharide storage reserve. This type of xyloglucan, which islocalized in massive thickenings on the inside of the seed cotyledoncell wall, is composed mainly of glucose, xylose and galactose (Rose etal., 1996).

Seeds of the tamarind tree Tamarindus indica became a commercial sourceof gum in 1943 when the gum was found useful as a paper and textilesize. Sizing of jute and cotton with tamarind xyloglucan has beenextensively practiced in Asia owing to the low cost of the gum and toits excellent properties. Food applications of tamarind xyloglucaninclude use in confections, jams and jellies and as a stabilizer in icecream and mayonnaise (Whistler et al., 1993).

Xyloglucanase activity is not included in the classification of enzymesprovided by the Enzyme Nomenclature (1992). Hitherto, this enzymaticactivity has simply been classified as glucanase activity and has oftenbeen believed to be identical to cellulolytic activity (EC 3.2.1.4).i.e., activity against beta-1,4-glycosidic linkages in cellulose orcellulose derivative substrates or at least to be a side activity inenzymes having cellulolytic activity. However, a true xyloglucanase is atrue xyloglucan specific enzyme capable of catalyzing the solubilizationof xyloglucan to xyloglucan oligosaccharides but which does not exhibitsubstantial cellulolytic activity, e.g., activity against theconventionally used cellulose-like substrates CMC(carboxymethylcellulose), HE cellulose and Avicel (microcrystallinecellulose). A xyloglucanase cleaves the beta-1,4-glycosidic linkages inthe backbone of xyloglucan.

Xyloglucanase activity is disclosed in Vincken et al., (1997) whereinthree different endoglucanases Endol, EndoV, and EndoVI from Trichodermaviride (similar to T. reesei) are characterized. Endol, EndoV and EndoVIbelong to families 5, 7 and 12 of glycosyl hydrolases, respectively, seeHenrissat, B. et al. (1991, 1993).

WO 94/14953 discloses a family 12 xyloglucanase (EG II) cloned from thefungus Aspergillus aculeatus and expressed in the fungus Aspergillisoryzae.

WO 99/02663 discloses xyloglucanases cloned from Bacillus licheniformis(family 12) and Bacillus agaradhaerens (family 5) and expressed inBacillus subtilis.

It is an object of the present invention to provide an enzyme with ahigh xyloglucanase activity at an alkaline pH which xyloglucanaseexhibits excellent performance in conventional detergent compositions.

SUMMARY OF THE INVENTION

The inventors have now found enzymes having substantial xyloglucanaseactivity, which enzymes belong to family 44 of glycosyl hydrolases andperform excellent in conventional detergent compositions, especially inliquid detergent compositions.

Accordingly, the present invention relates to an enzyme preparationcomprising a xyloglucanase belonging to family 44 of glycosyl hydrolasesand exhibiting a relative activity of at least 30% between pH 5.0 and8.0.

The inventors have also succeeded in cloning and expressing a family 44xyloglucanase. Accordingly, in further aspects the invention relates toa family 44 xyloglucanase which is (a) a polypeptide encoded by the DNAsequence of positions 121-1677 of SEQ ID NO: 1, (b) a polypeptideproduced by culturing a cell comprising the sequence of SEQ ID NO: 1under conditions wherein the DNA sequence is expressed, (c) axyloglucanase enzyme having a sequence of at least 60% identity topositions 40-559 of SEQ ID NO: 2 when identity is determined by GAPprovided in the GCG program package using a GAP creation penalty of 3.0and GAP extension penalty of 0.1, or (d) a polypeptide encoded by a DNAsequence that hybridizes to the DNA sequence of SEQ ID NO: 1 undermedium stringency conditions, wherein the medium stringency conditionscomprise hybridization in 5×SSC at 45° C. and washing in 2×SSC at 60°C.; or (e) a polypeptide encoded by the xyloglucanase encoding part ofthe DNA sequence obtainable from the plasmid in Escherichia coli DSM13321; and to a family 44 xyloglucanase which is (a) a polypeptideencoded by the DNA sequence of positions 121-1677 of SEQ ID NO: 3 (b) apolypeptide produced by culturing a cell comprising the sequence of SEQID NO: 3 under conditions wherein the DNA sequence is expressed, or (c)a polypeptide encoded by the xyloglucanase encoding part of the DNAsequence obtainable from the plasmid in Escherichia coil DSM 13322; andto a family 44 xyloglucanase which is (a) a polypeptide encoded by theDNA sequence of positions 121-1677 of SEQ ID NO: 5, (b) a polypeptideproduced by culturing a cell comprising the sequence of SEQ ID NO: 5under conditions wherein the DNA sequence is expressed, or (c) apolypeptide encoded by the xyloglucanase encoding part of the DNAsequence obtainable from the plasmid in Escherichia coli DSM 13323; andto an isolated polynucleotide molecule encoding a polypeptide havingxyloglucanase activity which polynucleotide molecule hybridizes to adenatured double-stranded DNA probe under medium stringency conditions,wherein the probe is selected from the group consisting of DNA probescomprising the sequence shown in positions 121-1677 of SEQ ID NO: 1, 3or 5, and DNA probes comprising a subsequence of positions 121-1677 ofSEQ ID NO: 1, 3 or 5, the subsequence having a length of at least about100 base pairs.

In further aspects, the invention provides an expression vectorcomprising a DNA segment which is, e.g., a polynucleotide molecule ofthe invention, a cell comprising the DNA segment or the expressionvector; and a method of producing a exhibiting xyloglucanase enzymewhich method comprises culturing the cell under conditions permittingthe production of the enzyme, and recovering the enzyme from theculture.

In yet another aspect the invention provides an isolated xyloglucanaseenzyme characterized in (i) being free from homologous impurities and(ii) being produced by the method described above.

The novel enzyme of the present invention is useful for the treatment ofcellulosic material, especially cellulose-containing fiber, yarn, wovenor non-woven fabric. The treatment can be carried out during theprocessing of cellulosic material into a material ready for garmentmanufacture or fabric manufacture, e.g., in the desizing or scouringstep; or during industrial or household laundering of such fabric orgarment.

Accordingly, in further aspects the present invention relates to adetergent composition comprising a xyloglucanase enzyme havingsubstantial xyloglucanase activity in the neutral or alkaline range; andto use of the enzyme of the invention for the treatment ofcellulose-containing fibers, yarn, woven or non-woven fabric.

The present invention has now made it possible to use a xyloglucanase indetergent compositions for removing or bleaching certain soils or stainspresent on laundry, especially soils and spots resulting fromxyloglucan-containing food, plants, and the like. Further, it iscontemplated that treatment with detergent compositions comprising thenovel enzyme can prevent binding of certain soils to the xyloglucan lefton the cellulosic material.

DETAILED DESCRIPTION OF THE INVENTION Microbial Sources

For the purpose of the present invention the term “obtained from” or“obtainable from” as used herein in connection with a specific source,means that the enzyme is produced or can be produced by the specificsource, or by a cell in which a gene from the source have been inserted.

It is at present contemplated that the xyloglucanase of the inventionmay be obtained from a gram-positive bacterium belonging to a strain ofthe genus Bacillus, in particular a strain of Paenibacillus.

In a preferred embodiment, the xyloglucanase of the invention isobtained from the strain Paenibacillus polymyxa, ATCC 832, which ispublicly available from American Type Culture Collection (ATCC). This isthe type strain of Paenibacillus polymyxa. It is at present contemplatedthat a DNA sequence encoding an enzyme with an amino acid sequenceidentity of at least 60% to the enzyme of the invention may be obtainedfrom other strains belonging to the genus Paenibacillus.

Further, the strain Paenibacillus sp. was deposited by the inventorsaccording to the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure at theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1b, D-38124 Braunschweig, Federal Republic of Germany, on 18 Feb.2000 under the deposition number DSM 13329. The deposit was made by NovoNordisk A/S and was later assigned to Novozymes A/S.

A plasmid comprising a DNA sequence encoding a xyloglucanase of theinvention has been transformed into a strain of the Escherichia coliwhich was deposited by the inventors according to the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, Federal Republic of Germany, on 16 Feb. 2000 under thedeposition number DSM 13321. The deposit was made by Novo Nordisk A/Sand was later assigned to Novozymes A/S. It is contemplated that the DNAsequence of this plasmid comprises the DNA sequence of SEQ ID NO: 1.

A plasmid comprising a DNA sequence encoding a xyloglucanase of theinvention has been transformed into a strain of the Escherichia coliwhich was deposited by the inventors according to the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, Federal Republic of Germany, on 16 Feb. 2000 under thedeposition number DSM 13322. The deposit was made by Novo Nordisk A/Sand was later assigned to Novozymes A/S. It is contemplated that the DNAsequence of this plasmid comprises the DNA sequence of SEQ ID NO: 3.

A plasmid comprising a DNA sequence encoding a xyloglucanase of theinvention has been transformed into a strain of the Escherichia coliwhich was deposited by the inventors according to the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, Federal Republic of Germany, on 16 Feb. 2000 under thedeposition number DSM 13323. The deposit was made by Novo Nordisk A/Sand was later assigned to Novozymes A/S. It is contemplated that the DNAsequence of this plasmid comprises the DNA sequence of SEQ ID NO: 5.

DEFINITIONS

In the present context, the term “enzyme preparation” is intended tomean either be a conventional enzymatic fermentation product, possiblyisolated and purified, from a single species of a microorganism, suchpreparation usually comprising a number of different enzymaticactivities; or a mixture of monocomponent enzymes, preferably enzymesderived from bacterial or fungal species by using conventionalrecombinant techniques, which enzymes have been fermented and possiblyisolated and purified separately and which may originate from differentspecies, preferably fungal or bacterial species; or the fermentationproduct of a microorganism which acts as a host cell for expression of arecombinant xyloglucanase, but which microorganism simultaneouslyproduces other enzymes, e.g., xyloglucanases proteases, or cellulases,being naturally occurring fermentation products of the microorganism,i.e., the enzyme complex conventionally produced by the correspondingnaturally occurring microorganism.

In the present context the term “expression vector” denotes a DNAmolecule, linear or circular, that comprises a segment encoding apolypeptide of interest operably linked to additional segments thatprovide for its transcription. Such additional segments may includepromoter and terminator sequences, and may optionally include one ormore origins of replication, one or more selectable markers, anenhancer, a polyadenylation signal, and the like. Expression vectors aregenerally derived from plasmid or viral DNA, or may contain elements ofboth. The expression vector of the invention may be any expressionvector that is conveniently subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which thevector is to be introduced. Thus, the vector may be an autonomouslyreplicating vector, i.e., a vector that exists as an extra chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid. Alternatively, the vector may be onewhich, when introduced into a host cell, is integrated into the hostcell genome and replicated together with the chromosome(s) into which ithas been integrated.

The term “recombinant expressed” or “recombinantly expressed” usedherein in connection with expression of a polypeptide or protein isdefined according to the standard definition in the art. Using anexpression vector as described immediately above generally performsrecombinant expression of a protein.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). The term “an isolated polynucleotide” mayalternatively be termed “a cloned polynucleotide”.

When applied to a protein/polypeptide, the term “isolated” indicatesthat the protein is found in a condition other than its nativeenvironment. In a preferred form, the isolated protein is substantiallyfree of other proteins, particularly other homologous proteins (i.e.,“homologous impurities” (see below)). It is preferred to provide theprotein in a greater than 40% pure form, more preferably greater than60% pure form.

Even more preferably it is preferred to provide the protein in a highlypurified form, i.e., greater than 80% pure, more preferably greater than95% pure, and even more preferably greater than 99% pure, as determinedby SDS-PAGE.

The term “isolated protein/polypeptide may alternatively be termed“purified protein/polypeptide”.

The term “homologous impurities” means any impurity (e.g., anotherpolypeptide than the polypeptide of the invention), which originate fromthe homologous cell where the polypeptide of the invention is originallyobtained.

The term “obtained from” as used herein in connection with a specificmicrobial source, means that the polynucleotide and/or polypeptideproduced by the specific source, or by a cell in which a gene from thesource have been inserted.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′-ATGCACGGG-3′ is complementary to 5-CCCGTGCAT-3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

Polynucleotides

Within preferred embodiments of the invention an isolated polynucleotideof the invention will hybridize to similar sized regions of SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 5, or a sequence complementary thereto,under at least medium stringency conditions.

In particular polynucleotides of the invention will hybridize to adenatured double-stranded DNA probe comprising either the full sequenceshown in SEQ ID NO: 1 or the sequence shown in positions 121-1677 of SEQID NO: 1 or the full sequence shown in SEQ ID NO: 3 or the sequenceshown in positions 121-1677 of SEQ ID NO: 3 or the partial sequenceshown in SEQ ID NO: 5 or the sequence shown in positions 121-1677 of SEQID NO: 5 or any probe comprising a subsequence of SEQ ID NO: 5 or SEQ IDNO: 3 or SEQ ID NO: 1 having a length of at least about 100 base pairsunder at least medium stringency conditions, but preferably at highstringency conditions as described in detail below. Suitableexperimental conditions for determining hybridization at medium or highstringency between a nucleotide probe and a homologous DNA or RNAsequence involve pre-soaking of the filter containing the DNA fragmentsor RNA to hybridize in 5×SSC (Sodium chloride/Sodium citrate, Sambrooket al., 1989) for 10 min, and prehybridization of the filter in asolution of 5×SSC, 5×Denhardt's solution (Sambrook et alt, 1989), 0.5%SDS and 100 micrograms/ml of denatured sonicated salmon sperm DNA(Sambrook et al., 1989), followed by hybridization in the same solutioncontaining a concentration of 10 ng/ml of a random-primed (Feinberg, A.P. and Vogelstein, B., 1983, Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity higher than 1×109 cpm/microgram) probe for 12 hoursat ca. 45° C. The filter is then washed twice for 30 minutes in 2×SSC,0.5% SDS at least 60° C. (medium stringency), still more preferably atleast 65° C. (medium/high stringency), even more preferably at least 70°C. (high stringency), and even more preferably at least 75° C. (veryhigh stringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an x-ray film.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the ar. DNA and RNA encoding genes of interest can becloned in Gene Banks or DNA libraries by means of methods known in theart.

Polynucleotides encoding polypeptides having endoglucanase activity ofthe invention are then identified and isolated by, for example,hybridization or PCR.

The present invention further provides counterpart polypeptides andpolynucleotides from different bacterial strains (orthologs orparalogs). Of particular interest are xyloglucanase polypeptides fromgram-positive alkalophilic strains, including species of Bacillus. Ofspecial interest are xyloglucanase peptides from strains which are veryclosely related to the strain Paenibacillus polymyxa, ATCC 832, which isthe type strain of Paenibacillus polymyxa.

Species homologues of a polypeptide with xyloglucanase activity of theinvention can be cloned using information and compositions provided bythe present invention in combination with conventional cloningtechniques. For example, a DNA sequence of the present invention can becloned using chromosomal DNA obtained from a cell type that expressesthe protein. Suitable sources of DNA can be identified by probingNorthern blots with probes designed from the sequences disclosed herein.A library is then prepared from chromosomal DNA of a positive cell line.A DNA sequence of the invention encoding an polypeptide havingxyloglucanase activity can then be isolated by a variety of methods,such as by probing with probes designed from the sequences disclosed inthe present specification and claims or with one or more sets ofdegenerate probes based on the disclosed sequences. A DNA sequence ofthe invention can also be cloned using the polymerase chain reaction, orPCR (Mullis, U.S. Pat. No. 4,683,202), using primers designed from thesequences disclosed herein. Within an additional method, the DNA librarycan be used to transform or transfect host cells, and expression of theDNA of interest can be detected with an antibody (monoclonal orpolyclonal) raised against the xyloglucanase cloned from Paenibacilluspolymyxa, e.g., from the type strain deposited as ATCC 832, or fromPaenibacillus sp., DSM 13329, expressed and purified as described inMaterials and Methods and Examples 1, 2 and 37 or by an activity testrelating to a polypeptide having xyloglucanase activity.

Polypeptides:

The sequence of amino acids 40-559 of SEQ ID NO: 2, 4 and 6,respectively, is a mature xyloglucanase sequence of the catalytic activedomain. The mature xyloglucanase sequence may in theory start atposition 35 or 36 but the enzyme as such has the amino acid sequencestarting at position 40 due to proteolytic maturing. Further, it shouldbe noted that the genes disclosed herein (SEQ ID NOS: 1, 3, 5) in totoencodes multidomain enzymes, i.e., encodes a xyloglucanase domain(catalytic active domain), a mannanase domain (catalytic a activedomain) and a binding domain. However, only the part of the genesencoding the xyloglucanase domain is relevant for the present invention.

The present invention also provides xyloglucanase polypeptides that aresubstantially homologous to the polypeptide of amino acids 40-559 of SEQID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and species homologs (paralogs ororthologs) thereof. The term “substantially homologous” is used hereinto denote polypeptides having 60%, preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, and even more preferably at least 90%, sequence identityto the sequence shown in amino acids 40-559 of SEQ ID NO: 2, 4 or 6 ortheir orthologs or paralogs. Such polypeptides will more preferably beat least 95% identical, and most preferably 98% or more identical to thesequence shown in amino acids 40-559 of SEQ ID NO: 2, 4 or 6 or itsorthologs or paralogs. Percent sequence identity is determined byconventional methods, by means of computer programs known in the artsuch as GAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) as disclosed in Needleman, S.B. and Wunsch, C. D., 1970, Journal of Molecular Biology, 48, 443-453,which is hereby incorporated by reference in its entirety. GAP is usedwith the following settings for polypeptide sequence comparison: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1. The followingsequence identity was found for the appended SEQ ID NOS: 2, 4 and 6;

SEQ ID #2 SEQ ID #4 SEQ ID #6 SEQ ID #2 100% 92% 84%

Sequence identity of polynucleotide molecules is determined by similarmethods using GAP with the following settings for DNA sequencecomparison; GAP creation penalty of 5.0 and GAP extension penalty of0.3.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 2) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tract, protein A (Nilsson etal., 1985. EMBO J. 4: 1075; Nilsson et al., 1991, Methods Enzymol. 198:3. See, in general Ford et at, 1991, Protein Expression and Purification2: 95-107, which is incorporated herein by reference. DNAs encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.).

However, even though the changes described above preferably are of aminor nature, such changes may also be of a larger nature such as fusionof larger polypeptides of up to 300 amino acids or more both as amino orcarboxyl-terminal extensions to a polypeptide of the invention havingxyloglucanase activity.

TABLE 1 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and a-methyl serine) may be substituted for amino acidresidues of a polypeptide according to the invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, or preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the xyloglucanase polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Weiss, 1989, Science 244: 1081-1085). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (i.e., xyloglucanase activity) to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction or photo affinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al, 1992,Science 255: 306-312; Smith et al., 1992, J. Mol. Bio. 224; 899-904:Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of homologieswith polypeptides, which are related to a polypeptide according to theinvention.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis, recombination and/or shuffling followed by arelevant screening procedure, such as those disclosed by Reidhaar-Olsonand Sauer, 1988, Science 241: 53-57), Bowie and Sauer, 1989, Proc. Natl.Acad. Sci. USA 86: 2152-2156, WO 95/17413, or WO 95/22625. Briefly,these authors disclose methods for simultaneously randomizing two ormore positions in a polypeptide, or recombination/shuffling of differentmutations (WO 95/17413, WO 95/22625), followed by selecting forfunctional a polypeptide, and then sequencing the mutagenizedpolypeptides to determine the spectrum of allowable substitutions ateach position. Other methods that can be used include phage display(e.g. Lowman et al., 1991, Biochem. 30: 10832-10837; Ladner et at, U.S.Pat. No. 5,223,409; Huse, WO 92/06204) and region-directed mutagenesis(Derbyshire et at, 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are substantiallyhomologous to residues 40 to 559 of SEQ ID NO: 2, 4 or 6 and retain thexyloglucanase activity of the wild-type protein.

The xyloglucanase enzyme of the invention may, in addition to the enzymecore comprising the catalytically domain, also comprise a cellulosebinding domain (CBD), the cellulose binding domain and enzyme core (thecatalytically active domain) of the enzyme being operably linked. Thecellulose-binding domain (CBD) may exist as an integral part the encodedenzyme, or a CBD from another origin may be introduced into thexyloglucanase thus creating an enzyme hybrid. In this context, the term“cellulose-binding domain” is intended to be understood as defined byPeter Tomme et at, “Cellulose-Binding Domains: Classification andProperties” in “Enzymatic Degradation of Insoluble Carbohydrates”, JohnN. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618,1996. This definition classifies more than 120 cellulose-binding domainsinto 10 families (I-X), and demonstrates that CBDs are found in variousenzymes such as cellulases, xylanases, mannanases, arabinofuranosidases,acetyl esterases and chitinases. CBDs have also been found in algae,e.g., the red alga Porphyra purpurea as a non-hydrolyticpolysaccharide-binding protein, see Tomme et al., op.cit. However, mostof the CBDs are from cellulases and xylanases, CBDs are found at the Nand C termini of proteins or are internal. Enzyme hybrids are known inthe art, see e.g., WO 90/00609 and WO 95/16782, and may be prepared bytransforming into a host cell a DNA construct comprising at least afragment of DNA encoding the cellulose-binding domain ligated, with orwithout a linker, to a DNA sequence encoding the xyloglucanase andgrowing the host cell to express the fused gene. Enzyme hybrids may bedescribed by the following formula.

CBD−MR−X

wherein CBD is the N-terminal or the C-terminal region of an amino acidsequence corresponding to at least the cellulose-binding domain; MR isthe middle region (the linker), and may be a bond, or a short linkinggroup preferably of from about 2 to about 100 carbon atoms, morepreferably of from 2 to 40 carbon atoms; or is preferably from about 2to about 100 amino acids, more preferably of from 2 to 40 amino acids;and X is an N-terminal or C-terminal region of a polypeptide encoded bythe polynucleotide molecule of the invention.

Immunological Cross-Reactivity

Polyclonal antibodies, especially monospecific polyclonal antibodies, tobe used in determining immunological cross-reactivity may be prepared byuse of a purified xyloglucanolytic enzyme. More specifically antiserumagainst the xyloglucanase of the invention may be raised by immunizingrabbits (or other rodents) according to the procedure described by N.Axelsen et al., in, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone andR. Thorpe, Immunochemistry in Practice, Blackwell ScientificPublications, 1982 (more specifically pp. 27-31). Purifiedimmunoglobulins may be obtained from the antisera, for example by saltprecipitation ((NH₄)₂SO₄), followed by dialysis and ion exchangechromatography, e.g., on DEAE-Sephadex. Immunochemical characterizationof proteins may be done either by Outcherlony double-diffusion analysis(O. Ouchterlony in; Handbook of Experimental Immunology (D. M. Weir,Ed.), Blackwell Scientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapters 3 and 4), orby rocket immunoelectrophoresis (N. Axelsen et al. Chapter 2).

Recombinant Expression Vectors

A recombinant vector comprising a DNA construct encoding the enzyme ofthe invention may be any vector, which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e., a vector that exists as anextra chromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome in part or in its entirety and replicated together withthe chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the enzyme of the invention is operably linked to additionalsegments required for transcription of the DNA. In general, theexpression vector is derived from plasmid or viral DNA, or may containelements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells includethe promoter of the Bacillus stearothermophilus maltogenic amylase gene,the Bacillus licheniformis alpha-amylase gene, the Bacillusamyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkalineprotease gene, or the Bacillus pumilus xylosidase gene, or the phageLambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

The DNA sequence encoding the enzyme of the invention may also, ifnecessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g., antibiotics like kanamycin,chloramphenicol, erythromycin, tetracycline, spectinomycin, or the like,or resistance to heavy metals or herbicides.

To direct an enzyme of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the presentenzyme, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, or to assemble these sequences bysuitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

Host Cells

The cloned DNA molecule introduced into the host cell may be eitherhomologous or heterologous to the host in question. If homologous to thehost cell, i.e., produced by the host cell in nature, it will typicallybe operably connected to another promoter sequence or, if applicable,another secretory signal sequence and/or terminator sequence than in itsnatural environment. The term “homologous” is intended to include a DNAsequence encoding an enzyme native to the host organism in question. Theterm “heterologous” is intended to include a DNA sequence not expressedby the host cell in nature. Thus, the DNA sequence may be from anotherorganism, or it may be a synthetic sequence.

The host cell into which the cloned DNA molecule or the recombinantvector of the invention is introduced may be any cell, which is capableof producing the desired enzyme and includes bacteria, yeast, fungi andhigher eukaryotic cells.

Examples of bacterial host cells which on cultivation are capable ofproducing the enzyme of the invention may be a gram-positive bacteriasuch as a strain of Bacillus, in particular Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus circulans, Bacillus coagulans,Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis andBacillus thuringiensis, a strain of Lactobacillus, a strain ofStreptococcus, a strain of Streptomyces, in particular Streptomyceslividans and Streptomyces murinus, or the host cell may be agram-negative bacteria such as a strain of Escherichia coli.

The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. e.g., Sambrook et al., supra).

When expressing the enzyme in bacteria such as Escherichia coli, theenzyme may be retained in the cytoplasm, typically as insoluble granules(known as inclusion bodies), or may be directed to the periplasmic spaceby a bacterial secretion sequence. In the former case, the cells arelysed and the granules are recovered and denatured after which theenzyme is refolded by diluting the denaturing agent. In the latter case,the enzyme may be recovered from the periplasmic space by disrupting thecells, e.g., by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

When expressing the enzyme in gram-positive bacteria such as a strain ofBacillus or a strain of Streptomyces, the enzyme may be retained in thecytoplasm, or may be directed to the extra cellular medium by abacterial secretion sequence.

Examples of a fungal host cell which on cultivation are capable ofproducing the enzyme of the invention is e.g., a strain of Aspergillusor Fusarium, in particular Aspergillus awamori, Aspergillus nidulans,Aspergillus niger, Aspergillis oryzae, and Fusarium oxysporum, and astrain of Trichoderma, preferably Trichoderma harzianum, Trichodermareesei and Trichoderma viride.

Fungal cells may be transformed by a process involving protoplastformation and transformation of the protoplasts followed by regenerationof the cell wall in a manner known per se. The use of a strain ofAspergillus as a host cell is described in EP 238 023 (Novo NordiskA/S), the contents of which are hereby incorporated by reference.

Examples of a host cell of yeast origin which on cultivation are capableof producing the enzyme of the invention is e.g., a strain of Hansenulasp., a strain of Kluyveromyces sp., in particular Kluyveromyces lactisand Kluyveromyces marcianus, a strain of Pichia sp., a strain ofSaccharomyces, in particular Saccharomyces carlsbergensis, Saccharomycescerevisae, Saccharomyces kluyveri and Saccharomyces uvarum, a strain ofSchizosaccharomyces sp., in particular Schizosaccharomyces pombe, and astrain of Yarrowia sp., in particular Yarrowia lipolytica.

Examples of host cells of plant origin which on cultivation are capableof producing the enzyme of the invention are e.g., a plant cell ofSolanum tuberosum or Nicotiana tabacum.

Method of Producing a Xyloglucanolytic Enzyme

In another aspect, the present invention also relates to a method ofproducing the enzyme preparation of the invention, the method comprisingculturing a microorganism capable of producing the xyloglucanase underconditions permitting the production of the enzyme, and recovering theenzyme from the culture. Culturing may be carried out using conventionalfermentation techniques, e.g., culturing in shake flasks or fermentorswith agitation to ensure sufficient aeration on a growth medium inducingproduction of the xyloglucanase enzyme. The growth medium may contain aconventional N-source such as peptone, yeast extract or casamino acids,a reduced amount of a conventional C-source such as dextrose or sucrose,and an inducer such as xyloglucan or composite plant substrates such ascereal bran (e.g., wheat bran or rice husk). The recovery may be carriedout using conventional techniques, e.g., separation of bio-mass andsupernatant by centrifugation or filtration, recovery of the supernatantor disruption of cells if the enzyme of interest is intracellular,perhaps followed by further purification as described in EP 0 406 314 orby crystallization as described in WO 97/15660.

Further, the present invention provides a method of producing anisolated enzyme according to the invention, wherein a suitable hostcell, which has been transformed with a DNA sequence encoding theenzyme, is cultured under conditions permitting the production of theenzyme, and the resulting enzyme is recovered from the culture.

As defined herein, an isolated polypeptide (e.g., an enzyme) is apolypeptide which is essentially free of other polypeptides, e.g., atleast about 20% pure, preferably at least about a 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

The term “isolated polypeptide” may alternatively be termed “purifiedpolypeptide”.

When an expression vector comprising a DNA sequence encoding the enzymeis transformed into a heterologous host cell it is possible to enableheterologous recombinant production of the enzyme of the invention.

Thereby it is possible to make a highly purified or monocomponentxyloglucanolytic composition, characterized in being free fromhomologous impurities.

In this present context “homologous impurities” means any impurities(e.g., other polypeptides than the enzyme of the invention), whichoriginate from the homologous cell where the enzyme of the invention isoriginally obtained.

In the present invention the homologous host cell may be a strain ofPaenibacillus sp. or Paenibacillus Polymyxa.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed xyloglucanolytic enzyme may conveniently be secreted into theculture medium and may be recovered there from by well-known proceduresincluding separating the cells from the medium by centrifugation orfiltration, precipitating proteinaceous components of the medium bymeans of a salt such as ammonium sulphate, followed by chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

The present invention also relates to a transgenic plant, plant part orplant cell which has been transformed with a DNA sequence encoding thexyloglucanase of the invention so as to express and produce this enzymein recoverable quantities. The enzyme may be recovered from the plant orplant part.

The transgenic plant can be dicotyledonous or monocotyledonous, forshort a dicot or a monocot. Examples of monocot plants are grasses, suchas meadow grass (blue grass, Poa), forage grass such as festuca, lolium,temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye,barley, rice, sorghum and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous (familyBrassicaceae), such as cauliflower, oil seed rape and the closelyrelated model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. In the present context, also specific plant tissues, such aschloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasmare considered to be a plant part. Furthermore, any plant cell, whateverthe tissue origin, is considered to be a plant part.

Also included within the scope of the invention are the progeny of suchplants, plant parts and plant cells.

The transgenic plant or plant cell expressing the enzyme of theinvention may be constructed in accordance with methods known in theart. In short the plant or plant cell is constructed by incorporatingone or more expression constructs encoding the enzyme of the inventioninto the plant host genome and propagating the resulting modified plantor plant cell into a transgenic plant or plant cell.

Conveniently, the expression construct is a DNA construct whichcomprises a gene encoding the enzyme of the invention in operableassociation with appropriate regulatory sequences required forexpression of the gene in the plant or plant part of choice. Furthermorethe expression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined,e.g., based on when, where and how the enzyme is desired to beexpressed. For instance, the expression of the gene encoding the enzymeof the invention may be constitutive or inducible, or may bedevelopmental, stage or tissue specific, and the gene product may betargeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are e.g., described by Tague et al., 1988, PlantPhys. 86: 506.

For constitutive expression the 35S-CaMV promoter may be used (Franck etat, 1980, Cell 21; 285-294). Organ-specific promoters may e.g. be apromoter from storage sink tissues such as seeds, potato tubers, andfruits (Edwards & Coruzzi, 1990, Annu. Rev Genet. 24, 275-303), or frommetabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol.Biol. 24: 863-878), a seed specific promoter such as the glutelin,prolamin, globulin or albumin promoter from rice (Wu et al. 1998, Plantand Cell Physiology 39(8). 885-889), a Vicia faba promoter from thelegumin B4 and the unknown seed protein gene from Vicia faba describedby Conrad U. et al., 1998, Journal of Plant Physiology 152(6): 708-711,a promoter from a seed oil body protein (Chen et al., 1998, Plant andCell Physiology 39(9): 935-941), the storage protein napA promoter fromBrassica napus, or any other seed specific promoter known in the art,e.g., as described in WO 91/14772. Furthermore, the promoter may be aleaf specific promoter such as the rbcs promoter from rice or tomato(Kyozuka et al., 1993, Plant Physiology 102(3); 991-1000), the chlorellavirus adenine methyltransferase gene promoter (Mitra, A. and Higgins, DW, 1994, Plant Molecular Biology 26(1); 8593), or the aldP gene promoterfrom rice (Kagaya et al., 1995, Molecular and General Genetics 248(6):668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Molecular Biology 22(4): 573-588).

A promoter enhancer element may be used to achieve higher expression ofthe enzyme in the plant. For instance, the promoter enhancer element maybe an intron placed between the promoter and the nucleotide sequenceencoding the enzyme. For instance, Xu et al., op cit, disclose the useof the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The DNA construct is incorporated into the plant genome according toconventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,micro injection, particle bombardment: biolistic transformation, andelectroporation (Gasser et al., Science 244: 1293; Potrykus, 1990,Bio/Techn. 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens mediated gene transfer is themethod of choice for generating transgenic dicots (for review Hooykas &Schilperoort, 1992, Plant Mol. Biol. 19: 15-38), however it can also beused for transforming monocots, although other transformation methodsare generally preferred for these plants. Presently, the method ofchoice for generating transgenic monocots is particle bombardment(microscopic gold or tungsten particles coated with the transformingDNA) of embryonic calli or developing embryos (Christou, 1992. Plant J.2: 275-281; Shimamoto, 1994, Curr Opin. Biotechnol 5: 158-162; Vasil etall, 1992. Bio/Technology 10: 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh S, et al., 1993, Plant Molecular Biology 21(3):415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art,

The Enzyme

In a preferred embodiment of the present invention, the xyloglucanasehas a relative activity at a temperature of 50° C., preferably of atleast 60%, preferably at least 70%, compared to the activity at theoptimal temperature.

In yet another preferred embodiment, at a temperature of 60° C., therelative xyloglucanase activity is at least 40%, preferably at least50%; at a temperature of 70° C., the relative xyloglucanase activity isat least 40%, preferably at least 45%, especially at least 50%.

Enzyme Compositions

In a still further aspect, the present invention relates to an enzymecomposition comprising an enzyme exhibiting xyloglucanase activity asdescribed above.

The enzyme composition of the invention may, in addition to thexyloglucanase of the invention, comprise one or more other enzyme types,for instance hemicellulase such as xylanase and mannanase, cellulase orendo-beta-1,4-glucanase components, chitinase, lipase, esterase,pectinase, cutinase, phytase, oxidoreductase (peroxidase,haloperoxidase, oxidase, laccase), protease, amylase, reductase,phenoloxidase, ligninase, pullulanase, pectate lyase, pectin acetylesterase, polygalacturonase, rhamnogalacturonase, pectin lyase, pectinmethylesterase, cellobiohydrolase, transglutaminase; or mixturesthereof.

The enzyme composition may be prepared in accordance with methods knownin the art and may be in the form of a liquid or a dry composition. Forinstance, the enzyme composition may be in the form of a granulate or amicrogranulate. The enzyme to be included in the composition may bestabilized in accordance with methods known in the art.

Xyloglucanases have potential uses in a lot of different industries andapplications. Examples are given below of preferred uses of the enzymecomposition of the invention. The dosage of the enzyme composition ofthe invention and other conditions under which the composition is usedmay be determined based on methods known in the art.

The xyloglucanase or xyloglucanase composition according to theinvention may be useful for at least one of the following purposes.

Uses Use in the Detergent Industry

During washing and wearing, dyestuff from dyed fabrics or garment willconventionally bleed from the fabric, which then looks faded and worn.Removal of surface fibers from the fabric will partly restore theoriginal colours and looks of the fabric. By the term “colourclarification”, as used herein, is meant the partly restoration of theinitial colours of fabric or garment throughout multiple washing cycles.

The term “de-pilling” denotes removing of pills from the fabric surface.

The term “soaking liquor” denotes an aqueous liquor in which laundry maybe immersed prior to being subjected to a conventional washing process.The soaking liquor may contain one or more ingredients conventionallyused in a washing or laundering process.

The term “washing liquor” denotes an aqueous liquor in which laundry issubjected to a washing process, i.e., usually a combined chemical andmechanical action either manually or in a washing machine.Conventionally, the washing liquor is an aqueous solution of a powder orliquid detergent composition.

The term “rinsing liquor” denotes an aqueous liquor in which laundry isimmersed and treated, conventionally immediately after being subjectedto a washing process, in order to rinse the laundry, i.e., essentiallyremove the detergent solution from the laundry. The rinsing liquor maycontain a fabric conditioning or softening composition.

The laundry subjected to the method of the present invention may beconventional washable laundry. Preferably, the major part of the laundryis sewn or unsown fabrics, including knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g., originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or rayon/viscose with one or more companion material such aswool, synthetic fibers (e.g., polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g., rayon/viscose,ramie, flax/linen, jute, cellulose acetate fibers, lyocell).

Detergent Disclosure and Examples Surfactant System

The detergent compositions according to the present invention comprise asurfactant system, wherein the surfactant can be selected from non-ionicand/or anionic and/or cationic and/or ampholytic and/or zwitterionicand/or semi-polar surfactants.

The surfactant is typically present at a level from 0.1% to 60% byweight.

The surfactant is preferably formulated to be compatible with enzymecomponents present in the composition. In liquid or gel compositions thesurfactant is most preferably formulated in such a way that it promotes,or at least does not degrade, the stability of any enzyme in thesecompositions.

Preferred systems to be used according to the present invention compriseas a surfactant one or more of the non-ionic and/or anionic surfactantsdescribed herein.

Polyethylene, polypropylene, and polybutylene oxide condensates of alkylphenols are suitable for use as the non-ionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available non-ionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkyl phenol alkoxylates(e.g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcoholswith about 1 to about 25 moles of ethylene oxide are suitable for use asthe non-ionic surfactant of the non-ionic surfactant systems of thepresent invention. The alkyl chain of the aliphatic alcohol can eitherbe straight or branched, primary or secondary, and generally containsfrom about 8 to about 22 carbon atoms. Preferred are the condensationproducts of alcohols having an alkyl group containing from about 8 toabout 20 carbon atoms, more preferably from about 10 to about 18 carbonatoms, with from about 2 to about 10 moles of ethylene oxide per mole ofalcohol. About 2 to about 7 moles of ethylene oxide and most preferablyfrom 2 to 5 moles of ethylene oxide per mole of alcohol are present insaid condensation products. Examples of commercially available non-ionicsurfactants of this type include Tergitol™ 15-S-9 (The condensationproduct of C₁₁-C₁₅ linear alcohol with 9 moles ethylene oxide),Tergitol™ 24-L-6 NMW (the condensation product of C₁₂-C₁₄ primaryalcohol with 6 moles ethylene oxide with a narrow molecular weightdistribution), both marketed by Union Carbide Corporation, Neodol™ 45-9(the condensation product of C₁₄-C₁₅ linear alcohol with 6 moles ofethylene oxide), Neodol™ 23-3 (the condensation product of C₁₂-C₁₃linear alcohol with 3.0 moles of ethylene oxide), Neodol™ 45-7 (thecondensation product of C₁₄-C₁₅ is linear alcohol with 7 moles ofethylene oxide), Neodol™ 45-5 (the condensation product of C₁₄-C₁₅linear alcohol with 5 moles of ethylene oxide) marketed by ShellChemical Company, Kyro™ EOB (the condensation of product of C₁₃-C₁₅alcohol with 9 moles ethylene oxide), marketed by The Procter & GambleCompany, and Genapol LA 050 (the condensation product of C₁₂-C₁₄ alcoholwith 5 moles of ethylene oxide) marketed by Hoechst. Preferred range ofHLB in these products is from 8-11 and most preferred from 8-10.

Also useful as the non-ionic surfactant of the surfactant systems of thepresent invention 1-% are alkyl polysaccharides disclosed in U.S. Pat.No. 4,565,647, having a hydrophobic group containing from about 6 toabout 30 carbon atoms, preferably from about 10 to about 16 carbon atomsand a polysaccharide, e.g., a polygclycoside, hydrophilic groupcontaining from about 1.3 to about 10, preferably from about 1.3 toabout 3, most preferably from about 1.3 to about 2.7 saccharide units.Any reducing saccharide containing 5 or 6 carbon atoms can be used,e.g., glucose, galactose and galactosyl moieties can be substituted forthe glycosyl moieties (optionally the hydrophobic group is attached atthe 2-, 3-, 4-, etc. positions thus giving a glucose or galactose asopposed to a glucoside or galactoside). The intersaccharide bonds canbe, e.g., between the one position of the additional saccharide unitsand the 2-, 3-, 4-, and/or 6-positions on the preceding saccharideunits.

The preferred alkyl polyglycosides have the formula

R²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)

wherein R² is selected from the group consisting of alkyd, alkyl phenyl,hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which thealkyl groups contain from about 10 to about 18, preferably from about 12to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 toabout 10, preferably 0; and x is from about 1.3 to about 10, preferablyfrom about 1.3 to about 3, most preferably from about 1.3 to about 2.7.The glycosyl is preferably derived from glucose. To prepare thesecompounds, the alcohol or alkylpolyethoxy alcohol is formed first andthen reacted with glucose, or a source of glucose, to form the glucoside(attachment at the 1-position. The additional glycosyl units can then beattached between their 1-position and the preceding glycosyl units 2-,3-, 4-, and/or 6-position, preferably predominantly the 2-position.

The condensation products of ethylene oxide with a hydrophobic baseformed by the condensation of propylene oxide with propylene glycol arealso suitable for use as the additional non-ionic surfactant systems ofthe present invention. The hydrophobic portion of these compounds willpreferably have a molecular weight from about 1500 to about 1800 andwill exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

Also suitable for use as the non-ionic surfactant of the non-ionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of non-ionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

Preferred for use as the non-ionic surfactant of the surfactant systemsof the present invention are polyethylene oxide condensates of alkylphenols condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄ alkylphenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₁₈ alcoholethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxy groups, andmixtures thereof.

Highly preferred non-ionic surfactants are polyhydroxy fatty acid amidesurfactants of the formula

wherein R¹ is H, or R¹ is C₁₋₄ hydroethyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxy hydrocarbyl having a linear hydrocarbyl chain with atleast 3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₆₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfatesurfactants. Examples hereof are water soluble salts or acids of theformula RO(A)_(m)SO3M wherein R is an a unsubstituted C₁₀-C₂₄ alkyl orhydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably C₁₂-C₂₀alkyl or hydroxyalkyl, more preferably C₁₂-C₁₈ alkyl or hydroxyalkyl, Ais an ethoxy or propoxy unit, m is greater than zero, typically betweenabout 0.5 and about 6, more preferably between about 0.5 and about 3,and M is H or a cation which can be, for example, a metal cation (e.g.,sodium, potassium, lithium, calcium, magnesium, etc.), ammonium orsubstituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkylpropoxylated sulfates are contemplated herein. Specific examples ofsubstituted ammonium cations include methyl, dimethyl,trimethyl-ammonium cations and quaternary ammonium cations such astetramethyl-ammonium and dimethyl piperidinium cations and those derivedfrom alkylamines such as ethylamine, diethyl amine, triethylamine,mixtures thereof, and the like. Exemplary surfactants are C₁₂-C₁₈ alkylpolyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈ alkylpolyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids), which are, sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 1975, 52: 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

The preferred alkyl ester sulfonate surfactant, especially for laundryapplications, comprises alkyl ester sulfonate surfactants of thestructural formula,

wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, or combinationthereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, or combinationthereof, and M is a cation, which forms a water-soluble salt with thealkyl ester sulfonate. Suitable salt-forming cations include metals suchas sodium, potassium, and lithium, and substituted or unsubstitutedammonium cations, such as monoethanolamine, diethanolamine, andtriethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl, and R⁴ is methyl,ethyl or isopropyl. Especially preferred are the methyl ester sulfonateswherein R³ is C₁₀-C₁₆ alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactantswhich are water soluble salts or acids of the formula ROSO₃M wherein Rpreferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl or hydroxyalkylhaving a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈ alkyl orhydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation(e.g., sodium, potassium, lithium), or ammonium or substituted ammonium(e.g., methyl, dimethyl, and trimethyl ammonium cations and quaternaryammonium cations such as tetramethyl-ammonium and dimethyl piperidiniumcations and quaternary ammonium cations derived from alkylamines such asethylamine, diethylamine, triethylamine, and mixtures thereof, and thelike). Typically, alkyl chains of C₁₂-C₁₆ are preferred for lower washtemperatures (e.g., below about 50° C.) and C₁₆-C₁₈ alkyl chains arepreferred for higher wash temperatures (e.g., above about 50° C.).

Other anionic surfactants useful for detersive purposes can also beincluded in the laundry detergent compositions of the present invention.Theses can include salts (including, for example, sodium, potassium,ammonium, and substituted ammonium salts such as mono-, di- andtriethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g. as described in British patent specification No.1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C₆-C₁₂ diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucosides (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH₂CH₂O)_(k)—CH₂COO-M+wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is asoluble salt forming cation. Resin acids and hydrogenated resin acidsare also suitable, such as rosin, hydrogenated rosin, and resin acidsand hydrogenated resin acids present in or derived from tall oil.

Alkylbenzene sulfonates are highly preferred. Especially preferred arelinear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkylgroup preferably contains from 10 to 18 carbon atoms.

Further examples are described in “Surface Active Agents and Detergents”(Vols. I and II by Schwartz, Perry and Berch). A variety of suchsurfactants are also generally disclosed in U.S. Pat. No. 3,929,678(Column 23, line 58 through Column 29, line 23, herein incorporated byreference).

When included therein, the laundry detergent compositions of the presentinvention typically comprise from about 1% to about 40%, preferably fromabout 3% to about 20% by weight of such anionic surfactants.

The laundry detergent compositions of the present invention may alsocontain cationic, ampholytic, zwitterionic, and semi-polar surfactants,as well as the nonionic and/or anionic surfactants other than thosealready described herein.

Cationic detersive surfactants suitable for use in the laundry detergentcompositions of the present invention are those having one long-chainhydrocarbyl group. Examples of such cationic surfactants include theammonium surfactants such as alkyltrimethylammonium halogenides, andthose surfactants having the formula:

[R²(OR³)_(y)][R⁴(OR³)_(y)]₂R⁵N+X−

wherein R² is an alkyl or alkyl benzyl group having from about 8 toabout 18 carbon atoms in the alkyl chain, each R³ is selected form thegroup consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—, —CH₂CH₂CH₂—,and mixtures thereof; each R⁴ is selected from the group consisting ofC₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structures formed byjoining the two R⁴ groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, wherein R⁶ is anyhexose or hexose polymer having a molecular weight less than about 1000,and hydrogen when y is not 0; R⁵ is the same as R⁴ or is an alkyl chain,wherein the total number of carbon atoms or R² plus R⁵ is not more thanabout 18; each y is from 0 to about 10, and the sum of the y values isfrom 0 to about 15; and X is any compatible anion.

Highly preferred cationic surfactants are the water soluble quaternaryammonium compounds useful in the present composition having the formula:

R₁R₂R₃R₄N⁺X⁻  (i)

wherein R₁ is C₈-C₁₆ alkyl, each of R₂, R₃ and R₄ is independentlyC₁-C₄alkyl, C₁-C₄ hydroxy alkyl benzyl, and —(C₂H₄₀)_(x)H where x has avalue from 2 to 5, and X is an anion. Not more than one of R₂, R₃ or R₄should be benzyl.

The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularly wherethe alkyl group is a mixture of chain lengths derived from coconut orpalm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

Preferred groups for R₂, R₃ and R₄ are methyl and hydroxyethyl groupsand the anion X may be selected from halide, methosulphate, acetate andphosphate ions.

Examples of suitable quaternary ammonium compounds of formula (i) foruse herein are,

coconut trimethyl ammonium chloride or bromide;coconut methyl dihydroxyethyl ammonium chloride or bromide;decyl triethyl ammonium chloride;decyl dimethyl hydroxyethyl ammonium chloride or bromide;C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;coconut dimethyl hydroxyethyl ammonium chloride or bromide;myristyl trimethyl ammonium methyl sulphate;lauryl dimethyl benzyl ammonium chloride or bromide;lauryl dimethyl(ethenoxy)₄ ammonium chloride or bromide;choline esters (compounds of formula (i) wherein R₁ is

di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in U.S. Pat.No. 4,228,044 and in EP 000 224.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 25%, preferably fromabout 1% to about 8% by weight of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight or branched-chain.One of the aliphatic substituents contains at least about 8 carbonatoms, typically from about 8 to about 18 carbon atoms, and at least onecontains an anionic water-solubilizing group, e.g., carboxy, sulfonate,sulfate. See U.S. Pat. No. 3,929,678 (column 19, lines 18-35) forexamples of ampholytic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergentcompositions. These surfactants can be broadly described as derivativesof secondary and tertiary amines, derivatives of heterocyclic secondaryand tertiary amines, or derivatives of quaternary ammonium, quaternaryphosphonium or tertiary sulfonium compounds. See U.S. Pat. No. 3,929,678(column 19, line 38 through column 22, line 48) for examples ofzwitterionic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such zwitterionic surfactants.

Semi-polar nonionic surfactants are a special category of nonionicsurfactants which include water-soluble amine oxides containing onealkyl moiety of from about 10 to about 18 carbon atoms and 2 moietiesselected from the group consisting of alkyl groups and hydroxyalkylgroups containing from about 1 to about 3 carbon atoms; watersolublephosphine oxides containing one alkyl moiety of from about 10 to about18 carbon atoms and 2 moieties selected from the group consisting ofalkyl groups and hydroxyalkyl groups containing from about 1 to about 3carbon atoms; and water-soluble sulfoxides containing one alkyl moietyfrom about 10 to about 18 carbon atoms and a moiety selected from thegroup consisting of alkyl and hydroxyalkyl moieties of from about 1 toabout 3 carbon atoms.

Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixturesthereof containing from about 8 to about 22 carbon atoms; R⁴ is analkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3; and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such semi-polar nonionic surfactants.

Builder System

The compositions according to the present invention may further comprisea builder 2$ system. Any conventional builder system is suitable for useherein including aluminosilicate materials, silicates, polycarboxylatesand fatty acids, materials such as ethylenediamine tetraacetate, metalion sequestrants such as aminopolyphosphonates, particularlyethylenediamine tetramethylene phosphonic acid and diethylene triaminepentamethylenephosphonic acid. Though less preferred for obviousenvironmental reasons, phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly aninorganic hydrated aluminosilicate material, more particularly ahydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, e.g.,SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting ofsodium silicate (Na₂Si₂O₅).

Suitable polycarboxylates containing one carboxy group include lacticacid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycolic acid, tartaric acid, tartronic acid and fumaric acid, as wellas the ether carboxylates described in German Offenleenschrift 2,446,686and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinyl carboxylatesdescribed in Belgian Patent No. 840,623. Polycarboxylates containingthree carboxy groups include, in particular, water-soluble citrates,aconitrates and citraconates as well as succinate derivatives such asthe carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinatesdisclosed in British Patent No. 1,261,829, 1,1,2,2,-ethanetetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfosubstituents include the sulfosuccinate derivatives disclosed in BritishPatent Nos. 1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, andthe sulfonated pyrolyzed citrates described in British Patent No.1,082,179, white polycarboxylates containing phosphono substituents aredisclosed in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates,2,3,4,5-tetrahydrofuran-cis,cis,cis-tetracarboxylates,2,5-tetrahydrofuran-cis-dicarboxylates,2,2,5,5-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexane-carboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are hydroxy-carboxylatescontaining up to three carboxy groups per molecule, more particularlycitrates.

Preferred builder systems for use in the present compositions include amixture of a water-insoluble aluminosilicate builder such as zeolite Aor of a layered silicate (SKS-6), and a water-soluble carboxylatechelating agent such as citric acid.

A suitable chalet for inclusion in the detergent compositions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

Other builder materials that can form part of the builder system for usein granular compositions include inorganic materials such as alkalimetal carbonates, bicarbonates, silicates, and organic materials such asthe organic phosphonates, amino polyalkylene phosphonates and aminopolycarboxylates.

Other suitable water-soluble organic salts are the homo- or co-polymericacids or their salts, in which the polycarboxylic acid comprises atleast two carboxyl radicals separated form each other by not more thantwo carbon atoms.

Polymers of this type are disclosed in GB-A-1,596,756. Examples of suchsalts are polyacrylates of MW 2,000-5,000 and their copolymers withmaleic anhydride, such copolymers having a molecular weight of from20,000 to 70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 5% to80% by weight of the composition. Preferred levels of builder for liquiddetergents are from 5% to 30%.

Enzymes

Preferred detergent compositions, in addition to the enzyme preparationof the invention, comprise other enzyme(s) which provides cleaningperformance and/or fabric care benefits.

Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g., laccases).

Proteases: Any protease suitable for use in alkaline solutions can beused. Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically or geneticallymodified mutants are included. The protease may be a serine protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisin, especially those derivedfrom Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).Examples of trypsin-like proteases are trypsin (e.g., of porcine orbovine origin) and the Fusarium protease described in WO 89/06270.

Preferred commercially available protease enzymes include those soldunder the trade names Alcalase, Savinase, Primase, Durazym, and Esperaseby Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase,Maxacal, Maxapem, Properase, Purafect and Purafect OXP by GenencorInternational, and those sold under the tradename Opticlean and Optimaseby Solvay Enzymes. Protease enzymes may be incorporated into thecompositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

Lipases: Any lipase suitable for use in alkaline solutions can be used.Suitable lipases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g.,as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase,e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., 1993, Biochemica etBiophysica Acta 1131: 253-260), a B. stearothermophilus lipase (JP641744992) and a B. pumilus lipase (WO 91/16422).

Furthermore, a number of cloned lipases may be useful, including thePenicillium camembertii lipase described by Yamaguchi et al., 1991, Gene103: 61-67), the Geotricum candidum lipase (Schimada, Y. et al., 1989,J. Biochem, 1068: 383-388), and various Rhizopus lipases such as a R.delemar lipase (Hass, M. J et at, 1991, Gene 109: 117-113), a R. niveuslipase (Kugimiya et at, 1992, Biosci. Biotech. Biochem. 56: 716-719) anda R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful,e.g., a cutinase derived from Pseudomonas mendocina as described in WO88/09367, or a cutinase derived from Fusarium solani pisi (e.g.,described in WO 90/09446).

Especially suitable lipases are lipases such as M1 Lipase™, Luma fast™and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (Novo NordiskA/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

The lipases are normality incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Amylases: Any amylase (alpha and/or beta) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, a-amylases obtained from a special strainof B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™(available from Genencor).

The amylases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Cellulases: Any cellulose suitable for use in alkaline solutions can beused. Suitable cellulases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307 which disclosesfungal cellulases produced from Humicola insolens, in WO 96/34108 and WO96/34092 which disclose bacterial alkalophilic cellulases (BCE 103) fromBacillus, and in WO 94/21801, U.S. Pat. No. 5,475,101, and U.S. Pat. No.5,419,778 which disclose EG III cellulases from Trichoderma. Especiallysuitable cellulases are the cellulases having colour care benefits.Examples of such cellulases are cellulases described in European patentapplication No. 0 495 257. Commercially available cellulases includeCelluzyme™ and Carezyme™ produced by a strain of Humicola insolens (NovoNordisk A/S), KAC-500(B)™ (Kao Corporation), and Puradax™ (GenencorInternational).

Cellulases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Peroxidases/Oxidases: Peroxidase enzymes are used in combination withhydrogen peroxide or a source thereof (e.g., a percarbonate, perborateor persulfate). Oxidase enzymes are used in combination with oxygen.Both types of enzymes are used for “solution bleaching”, i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in e.g., WO 94/12621 andWO 95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

Peroxidase and/or oxidase enzymes are normally incorporated in thedetergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a protease, an amylase, a lipase and/or acellulase.

The enzyme of the invention, or any other enzyme incorporated in thedetergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

Bleaching Agents

Additional optional detergent ingredients that can be included in thedetergent compositions of the present invention include bleaching agentssuch as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present, oxygen bleaching compounds willtypically be present at levels of from about 1% to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g., granular detergents.

The bleaching agent component for use herein can be any of the bleachingagents useful for detergent compositions including oxygen bleaches aswell as others known in the art.

The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

One category of oxygen bleaching agent that can be used encompassespercarboxylic acid bleaching agents and salts thereof. Suitable examplesof this class of agents include magnesium monoperoxyphthalatehexahydrate, the magnesium salt of meta-chloro perbenzoic acid,4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid.Such bleaching agents are disclosed in U.S. Pat. Nos. 4,412,934,4,483,781, and 4,740,446 and EP 0 133 354. Highly preferred bleachingagents also include 6-nonylamino-6-oxoperoxycaproic acid as described inU.S. Pat. No. 4,634,551.

Another category of bleaching agents that can be used encompasses thehalogen bleaching agents. Examples of hypohalite bleaching agents, forexample, include trichloro isocyanuric acid and the sodium and potassiumdichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.Such materials are normally added at 0.5-10% by weight of the finishedproduct, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination withbleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators Ca (6-octanamido-caproyl)oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

Useful bleaching agents, including peroxyacids and bleaching systemscomprising bleach activators and peroxygen bleaching compounds for usein cleaning compositions according to the invention are described inU.S. application no. 08/136,626.

The hydrogen peroxide may also be present by adding an enzymatic system(i.e., an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EP 0 537381.

Bleaching agents other than oxygen bleaching agents are also known inthe art and can be utilized herein. One type of non-oxygen bleachingagent of particular interest includes photoactivated bleaching agentssuch as the sulfonated zinc and/or aluminium phthalocyanines. Thesematerials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

Bleaching agents may also comprise a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in “Efficientmanganese catalysts for low-temperature bleaching”, 1994, Nature 369:637-639.

Suds Suppressors

Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwaterdispersible, substantially non surface-active detergent impermeablecarrier. Alternatively the suds suppressor can be dissolved or dispersedin a liquid carrier and applied by spraying on to one or more of theother components.

A preferred silicone suds controlling agent is disclosed in U.S. Pat.No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

Such suds suppressor systems are described in EP 0 593 841.

Especially preferred silicone suds controlling agents are described inEuropean Patent Application No. 92201649.8. Said compositions cancomprise a silicone/silica mixture in combination with fumed nonporoussilica such as Aerosil®.

The suds suppressors described above are normally employed at levels offrom 0.001% to 2% by weight of the composition, preferably from 0.01% to1% by weight.

Other Components

Other components used in detergent compositions may be employed such assoil-suspending agents, soil-releasing agents, optical brighteners,abrasives, bactericides, tarnish inhibitors, coloring agents, and/orencapsulated or nonencapsulated perfumes.

Especially suitable encapsulating materials are water soluble capsuleswhich consist of a matrix of polysaccharide and polyhydroxy compoundssuch as described in GB 1,464,616.

Other suitable water soluble encapsulating materials comprise dextrinsderived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

Antiredeposition and soil suspension agents suitable herein includecellulose derivatives such as methylcellulose, carboxymethylcelluloseand hydroxyethylcellulose, and homo- or co-polymeric polycarboxylicacids or their salts. Polymers of this type include the polyacrylatesand maleic anhydride-acrylic acid copolymers previously mentioned asbuilders, as well as copolymers of maleic anhydride with ethylene,methylvinyl ether or methacrylic acid, the maleic anhydride constitutingat least 20 mote percent of the copolymer. These materials are normallyused at levels of from 0.5% to 10% by weight, more preferably form 0.75%to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples ofwhich are disodium4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,disodium4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,monosodium4′,4″-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2-sulphonate,disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,disodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′-disulphonate,disodium4,4′-bis-(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,sodium 2-stilbene-4″-(naphtho-1′,2′:4,5)-1,2,3-triazole-2″-sulphonateand 4,4′-bis-(2-sulphostyrlyl)biphenyl.

Other useful polymeric materials are the polyethylene glycols,particularity those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and dealing performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention areconventionally copolymers or terpolymers of terephthalic acid withethylene glycol and/or propylene glycol units in various arrangements.Examples of such polymers are disclosed in U.S. Pat. Nos. 4,116,885 and4,711,730 and EP 0 272 033. A particular preferred polymer in accordancewith EP 0 272 033 has the formula,

(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25)[T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)

where PEG is —(OC₂H₄)O—, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

The selected polyesters herein contain about 46% by weight of dimethylterephthalic acid, about 16% by weight of 1,2-propanediol, about 10% byweight ethylene glycol, about 13% by weight of dimethyl sulfobenzoicacid and about 15% by weight of sulfoisophthalic acid, and have amolecular weight of about 3.000. The polyesters and their method ofpreparation are described in detail in EP 311 342.

Softening Agents

Fabric softening agents can also be incorporated into laundry detergentcompositions in accordance with the present invention. These agents maybe inorganic or organic in type. Inorganic softening agents areexemplified by the smectite clays disclosed in GB-A-1 400898 and in U.S.Pat. No. 5,019,292. Organic fabric softening agents include the waterinsoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0 011 340and their combination with mono C₁₂-C₁₄ quaternary ammonium salts aredisclosed in EP 0 026 528 and di-long-chain amides as disclosed in EP 0242 919. Other useful organic ingredients of fabric softening systemsinclude high molecular weight polyethylene oxide materials as disclosedin EP 0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 5% to 15%, morepreferably from 8% to 12% by weight, with the material being added as adry mixed component to the remainder of the formulation. Organic fabricsoftening agents such as the water-insoluble tertiary amines or di-longchain amide materials are incorporated at levels of from 0.5% to 5% byweight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

Polymeric Dye-Transfer Inhibiting Agents

The detergent compositions according to the present invention may alsocomprise from 0.001% to 10%, preferably from 0.01% to 2%, morepreferably form 0.05% to 1% by weight of polymeric dye transferinhibiting agents Said polymeric dye-transfer inhibiting agents arenormally incorporated into detergent compositions in order to inhibitthe transfer of dyes from colored fabrics onto fabrics washed therewith.These polymers have the ability of complexing or adsorbing the fugitivedyes washed out of dyed fabrics before the dyes have the opportunity tobecome attached to other articles in the wash.

Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

Addition of such polymers also enhances the performance of the enzymesaccording the invention.

The detergent composition according to the invention can be in liquid,paste, gels, bars or granular forms.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1,483,591.

Granular compositions according to the present invention can also be in“compact form”, i.e., they may have a relatively higher density thanconventional granular detergents, i.e., from 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

The compositions of the invention may for example, be formulated as handand machine laundry detergent compositions including laundry additivecompositions and compositions suitable for use in the pretreatment ofstained fabrics, rinse added fabric softener compositions, andcompositions for use in general household hard surface cleaningoperations and dishwashing operations.

The following examples are meant to exemplify compositions for thepresent invention, but are not necessarily meant to limit or otherwisedefine the scope of the invention.

In the detergent compositions, the abbreviated component identificationshave the following meanings:

LAS: Sodium linear C₁₂ alkyl benzene sulphonateTAS: Sodium tallow alkyl sulphateXYAS: Sodium C_(1X)-C_(1Y) alkyl sulfateSS: Secondary soap surfactant of formula 2-butyl octanoic acid25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensed with anaverage of Y moles of ethylene oxide25EY: A C₁₄-C₁₅ predominantly linear primary alcohol condensed with anaverage of Y moles of ethylene oxideXYEZS: C_(1X)-C_(1Y) sodium alkyl sulfate condensed with an average of Zmoles of ethylene oxide per moleNonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with anaverage degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbhCFAA: C₁₂-C₁₄ alkyl N-methyl glucamideTFAA: C16-C₁₈ alky N-methyl glucamideSilicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)NaSKS-6: Crystalline layered silicate of formula d—Na₂Si₂O₅Carbonate: Anhydrous sodium carbonatePhosphate: Sodium tripolyphosphateMA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weightabout 80,000Polyacrylate: Polyacrylate homopolymer with an average molecular weightof 8,000 sold under the tradename PA30 by BASF GmbHZeolite A: Hydrated Sodium Aluminosilicate of formula Na₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range from 1 to 10micrometersCitrate: Tri-sodium citrate dihydrate

Citric: Citric Acid

Perborate: Anhydrous sodium perborate monohydrate bleach, empiricalformula

NaBO₂.H₂O₂

PB4: Anhydrous sodium perborate tetrahydratePercarbonate: Anhydrous sodium percarbonate bleach of empirical formula2Na₂CO₃.3H₂O₂TAED: Tetraacetyl ethylene diamineCMC: Sodium carboxymethyl celluloseDETPMP: Diethylene triamine penta(methylene phosphonic acid), marketedby Monsanto under the Tradename Dequest 2060PVP: Polyvinylpyrrolidone polymerEDDS: Ethylenediamine-N,N′-disuccinic acid, [S,S] isomer in the form ofthe sodium saltSuds Suppressor: 25% paraffin wax Mpt 50° C., 17% hydrophobic silica,58% paraffin oilGranular Suds suppressor: 12% Silicone/silica, 18% stearyl alcohol, 70%starch in granular formSulphate: Anhydrous sodium sulphateHMWPEO: High molecular weight polyethylene oxideTAE 25: Tallow alcohol ethoxylate (25)

Detergent Example I

A granular fabric cleaning composition in accordance with the inventionmay be prepared as follows:

Sodium linear C₁₂ alkyl 6.5 benzene sulfonate Sodium sulfate 15.0Zeolite A 26.0 Sodium nitrilotriacetate 5.0 Enzyme of the invention 0.1PVP 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0 Phenol sulphonate 0.1Minors Up to 100

Detergent Example II

A compact granular fabric cleaning composition (density 800 g/l) inaccord with the invention may be prepared as follows:

45AS 8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-612.0 Citric acid 3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of theinvention 0.1 TAED 6.0 Percarbonate 22.0 EDDS 0.3 Granular sudssuppressor 3.5 water/minors Up to 100%

Detergent Example III

Granular fabric cleaning compositions in accordance with the inventionwhich are especially useful in the laundering of coloured fabrics wereprepared as follows:

LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS 3.1 10.0 45E7 4.0 — 25E3S 3.0 68E111.8 — 25E5 — 8.0 Citrate 15.0 7.0 Carbonate — 10 Citric acid 2.5 3.0Zeolite A 32.1 25.0 Na-SKS-6 — 9.0 MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzymeof the invention 0.10 0.05 Silicate 2.5 — Sulphate 5.2 3.0 PVP 0.5 —Poly (4-vinylpyridine)-N- — 0.2 Oxide/copolymer of vinyl- imidazole andvinyl- pyrrolidone Perborate 1.0 — Phenol sulfonate 0.2 — Water/MinorsUp to 100%

Detergent Example IV

Granular fabric cleaning compositions in accordance with the inventionwhich provide “Softening through the wash” capability may be prepared asfollows:

45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 — 5.0Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate 5.03.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

Detergent Example V

Heavy duty liquid fabric cleaning compositions in accordance with theinvention may be prepared as follows:

I II LAS acid form — 25.0 Citric acid 5.0 2.0 25AS acid form 8.0 —25AE2S acid form 3.0 — 25AE7 8.0 — CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8— Oleic acid — 1.0 Ethanol 4.0 6.0 Propanediol 2.0 8.0 Enzyme of theinvention 0.10 0.05 Coco-alkyl dimethyl — 3.0 hydroxy ethyl ammoniumchloride Smectite clay — 5.0 PVP 2.0 — Water/Minors Up to 100%

The Xyloglucan Substrate

In addition to the aforesaid about xyloglucan it should be noted thatxyloglucan from tamarind seeds supplied by Megazyme, Ireland has acomplex branched structure with glucose, xylose, galactose and arabinosein the ratio of 45:36:16:3. Accordingly, it is strongly believed that anenzyme showing catalytic activity on this xyloglucan also has catalyticactivity on other xyloglucan structures from different sources(angiosperms or gymnosperms).

Cotton suspension culture xyloglucan MW 100,000 kDa was obtained fromProfessor A. Mort of Oklahoma State University. 1H NMR (D2O, 80° C.) ofxyloglucans was used to compare the monosaccharide composition ofsamples of different origin. The integrals of the anomeric signals fromthe commercial sample fully agree with the composition given byMegazyme. However, the cotton xyloglucan seems to have a differentstructure. There appears to be much less galactose and about half ofgalactose residues are fucosylated. Furthermore, the molar ratio betweenxylose and glucose is smaller (0.63 compared to 0.77 for the tamarind),which suggest a more open structure of cotton xyloglucan. These findingsagree with results obtained with xyloglucan from cotton cells (Buchalaet al., 1993, Acta Bot. Neerl. 42; 213-219).

Xyloglucan (Megazyme) Cotton xyloglucan Glucose 45% 52% Xylose 35% 33%Galactose 16% 10% Fucose —  5% Arabinose   <4% a — a Could not bedetected in NMR

Materials and Methods Strains

Paenibacillus polymyxa, e.g., Paenibacillus polymyxa, ATCC 832, andPaenibacillus sp., DSM 13329, comprises a DNA sequence encoding axyloglucanase of the invention.

Other Strains

E. coli hosts: XL1-Blue MRF⁻ and XLOLR E. coli strains were provided byStratagene Inc. (USA) and used according to the manufacturer'sinstructions.

B. subtilis PL1885 (Diderichsen et al., 1990).

B. subtilis PL1801. This strain is B. subtilis DN1885 where the twomajor proteases have been inactivated (Diderichsen et al., 1990).

Competent cells were prepared and transformed as described by Yasbin etal. (1975).

Plasmids

pBK-CAMV. Stratagene Inc., La Jolla Calif., USA.

Bacteriophage ZAP Express: Stratagene Inc., La Jolla Calif., USA.

pMOL944. This plasmid is a pUB110 derivative essentially containingelements making the plasmid propagatable in Bacillus subtilis, kanamycinresistance gene and having a strong promoter and signal peptide clonedfrom the amyL gene of B. licheniformis ATCC 14580. The signal peptidecontains a SacII site making it convenient to clone the DNA encoding themature part of a protein in-fusion with the signal peptide. This resultsin the expression of a Pre-protein, which is directed towards theexterior of the cell.

The plasmid was constructed by means of ordinary genetic engineering andis briefly described in the following,

Construction of pMOL944:

The pUB110 plasmid (McKenzie, T. et al., 1986) was digested with theunique restriction enzyme Ncil. A PCR fragment amplified from the amyLpromoter encoded on the plasmid pDN1981 (Jørgensen et al., 1990) wasdigested with Ncil and inserted in the Ncil digested pUB110 to give theplasmid pSJ2624.

The two PCR primers used have the following sequences:

(SEQ ID NO: 7) # LWN54945′-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC-3′ (SEQ ID NO: 8)# LWN5495 5′-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAATGAGGCAGCAAGAAGAT-3′

The primer #LWN5494 inserts a NotI site in the plasmid.

The plasmid pSJ2624 was then digested with SacI and NotI and a new PCRfragment amplified on amyL promoter encoded on the pDN1981 was digestedwith SacI and NotI and this DNA fragment was inserted in the SacI-NotIdigested pSJ2624 to give the plasmid pSJ2670.

This cloning replaces the first amyL promoter cloning with the samepromoter but in the opposite direction. The two primers used for PCRamplification have the following sequences:

(SEQ ID NO: 9) #LWN59385′-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATG AGGCAGCAAGAAGAT-3′(SEQ ID NO: 10) #LWN5939 5′-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC-3′

The plasmid pSJ2670 was digested with the restriction enzymes PstI andBcII and a PCR fragment amplified from a cloned DNA sequence encodingthe alkaline amylase SP722 (WO 95/26397 which is hereby incorporated byreference) was digested with PstI and BcII and inserted to give theplasmid pMOL944. The two primers used for PCR amplification have thefollowing sequence:

(SEQ ID NO: 11) #LWN7864 5′-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3′ (SEQID NO: 12) #LWN7901 5′-AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG-3′

The primer #LWN7901 inserts a SacII site in the plasmid.

pPL3143: This plasmid is a pMol944 derivative in which a terminator hasbeen inserted between the SacII and the NotI site in pMOL944. At thesame time a new restriction site for cloning AscI has been inserted.

The plasmid was constructed by means of ordinary genetic engineering andis briefly described in the following.

Construction of pPL3143: The plasmid pMOL944 was digested with SacII andNotI. A PCR fragment generating a terminator was made using the twoprimers listed below and plasmid pMOL944 as template. This fragment wasdigested with EagI and SacII and inserted between the SacII and the NotIsite in PMOL944 to create the plasmid pPL3143.

Primer 130721: (SEQ ID NO: 13)5′-CGATCGGCCGATAAAAAAACCGGGCGGAAACCGCCCGTCATCTGGCGCGCCTATATACCGCGGCTGCAGAATGAGGCAGCAAG-3′ Primer 130722: (SEQ ID NO: 14)5′-GGCGCATTAACGGAATAAAGGGTGT-3′

Media

TY (as described in Ausubel, F. M. et al., 1995).

LB agar (as described in Ausubel, F. M. et al., 1995).

LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassiumphosphate, pH 7.0

AZCL-xyloglucan is added to LBPG-agar to 0.5%. AZCL-xyloglucan is fromMegazyme, Ireland.

BPX media is described in EP 0 506 780 (WO 91/09129).

NZY agar (per liter) 5 g of NaCl, 2 g of MgSO₄, 5 g of yeast extract 10g of NZ amine (casein hydrolysate), 15 g of agar, add deionized water to1 titer, adjust pH with NaOH to pH 7.5 and autoclave.

NZY broth (per liter) 5 g of NaCl, 2 g of MgSO₄, 5 g of yeast extract,10 g of NZ amine (casein hydrolysate); add deionized water to 1 liter,adjust pH with NaOH to pH 7.5 and autoclave.

NZY Top Agar (per liter) 5 g of NaCl, 2 g of MgSO₄, 5 g of yeastextract, 10 g of NZ amine (casein hydrolysate), 0.7% (w/v) agarose; adddeionized water to 1 liter, adjust pH with NaOH to pH 7.5 and autoclave.

Xyloglucanase assay (XyloU)

The xyloglucanase activity is measured using AZCL-xyloglucan fromMegazyme, Ireland, (megazyme.com/purchase/index.html) as substrate.

A solution of 0.2% of the blue substrate is suspended in a 0.1 Mphosphate buffer pH 7.5 under stirring. The solution is distributedunder stirring to 1.5 ml Eppendorf tubes (0.75 ml to each), 50microliters enzyme solution is added and they are incubated in anEppendorp Thermomixer model 5436 for 20 minutes at 40° C. with a mixingof 1200 rpm. After incubation the colored solution is separated from thesolid by 4 minutes centrifugation at 14,000 rpm and the absorbance ofthe supernatant is measured at 600 nm.

One XyloU unit is defined as the amount of enzyme resulting in anabsorbance of 0.24 in a 1 cm cuvette at 600 nm.

General Molecular Biology Methods

DNA manipulations and transformations were performed using standardmethods of molecular biology (Sambrook et al., 1989, Molecular cloning:A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.;Ausubel, F. M. et al. (eds.), “Current Protocols in Molecular Biology”,John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)“Molecular Biological Methods for Bacillus,” John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the specificationsof the suppliers.

The following examples illustrate the invention.

EXAMPLE 1 Cloning of Xyloglucanase Encoding Genes from PaenibacillusSpecies Genomic DNA Operation

Strains of Paenibacillus polymyxa, including Paenibacillus polymyxa,ATCC 842, and the strain Paenibacillus sp., DSM 13329, respectively,were propagated in liquid TY medium. After 16 hours incubation at 30° C.and 300 rpm, the cells were harvested, and genomic DNA isolated by themethod described by Pitcher et al. (1989).

Genomic Library Construction

Lambda ZAP libraries were prepared from genomic DNA of the strainsPaenibacillus polymyxa, Paenibacillus polymyxa. ATCC 842, Paenibacillussp., DSM 13329. The ZAP Express cloning kit used was with BamHI digestedand dephosphorylated arms from Stratagene. Isolated DNA was partiallydigested with Sau3A and size fractionated on a 1% agarose gel, DNA wasexcised from the agarose gel between 3 and 10 Kb and purified usingQiaspin DNA fragment purification procedure (Qiagen GmbH). 100 ng ofpurified, fractionated DNA was ligated with 1 microgram of BamHIdephosphorylated ZAPexpress vector arms (4 degrees overnight). Ligationreaction was packaged directly with GigaPackIII Gold according to themanufacturer's instructions (Stratagene). Phage libraries were titeredwith XL1 blue mrf (Stratagene).

Screening for Xyloglucanase Clones by Functional Expression inLambdaZAPExpress

Approximately 10,000 plaque-forming units (pfu) from the genomic librarywere plated on NZY-agar plates containing 0.1% AZCL-xyloglucan(MegaZyme, Ireland), using E. coli XL1-Blue MRF' (Stratagene, USA) as ahost, followed by incubation of the plates at 37° C. for 24 hours.Xyloglucanase-positive lambda clones were identified by the formation ofblue hydrolysis halos around the positive phage clones. These wererecovered from the screening plates by coring the TOP-agar slicescontaining the plaques of interest into 500 microliters of SM buffer and20 microliters of chloroform. The xyloglucanase-positivelambdaZAPExpress clones were plaque-purified by plating an aliquot ofthe cored phage stock on NZY plates containing 0.1% AZCL-xyloglucan asabove. Single xyloglucanase-positive lambda clones were cored into 500microliters of SM buffer and 20 microliters of chloroform, and purifiedby one more plating round as described above.

Single-Clone In Vivo Excision of the Phagemids from theXyloglucanase-Positive LambdaZAPExpress clones

E. coli XL1-Blue cells (Stratagene, La Jolla Calif.) were prepared andresuspended in 10 mM MgSO₄ as recommended by Stratagene (La Jolla, USA).250 microliter aliquots of the pure phage stocks from thexyloglucanase-positive clones were combined in Falcon 2059 tubes with200 microliters of XL1-Blue MRF' cells (OD600=1.0) and >10⁶ pfu/ml ofthe ExAssist M13 helper phage (Stratagene), and the mixtures wereincubated at 37° C. for 15 minutes. 3 ml of NZY broth was added to eachtube and the tubes were incubated at 37° C. for 2.5 hours. The tubeswere heated at 65° C. for 20 minutes to kill the E. coli cells andbacteriophage lambda; the phagemids being resistant to heating. Thetubes were spun at 3000 rpm for 15 minutes to remove cellular debris andthe supernatants were decanted into clean Falcon 2059 tubes. Aliquots ofthe supernatants containing the excised single-stranded phagemids wereused to infect 200 microliters of E. coli XLOLR cells (Stratagene,OD600=1.0 in 10 mM MgSO₄) by incubation at 37° C. for 15 minutes. 350microliters of NZY broth was added to the cells and the tubes wereincubated for 45 min at 37° C. Aliquots of the cells were plated onto LBkanamycin agar plates and incubated for 24 hours at 37° C. Five excisedsingle colonies were re-streaked onto LB kanamycin agar platescontaining 0.1% AZCL-xyloglucan (MegaZyme, Australia). Thexyloglucanase-positive phagemid clones were characterized by theformation of blue hydrolysis halos around the positive colonies. Thesewere further analysed by restriction enzyme digests of the isolatedphagemid DNA (QiaSpin kit, Qiagen, USA) with EcoRI, PstI, EcoRI-PstI,and HindIII followed by agarose gel electrophoresis.

Nucleotide Sequence Analysis

The nucleotide sequence of the genomic xyloglucanase clones weredetermined from both strands by the dideoxy chain-termination method(Sanger et al, 1977) using 500 ng of Qiaspin-purified template (Qiagen,USA), the Taq deoxy-terminal cycle sequencing kit (Perkin-Elmer, USA),fluorescent labelled terminators and 5 Pmol of either pBK-CMV polylinkerprimers (Stratagene, USA) or custom synthetic oligonucleotide primers.DNA sequence assembly was performed with the DNA Star package(DNASTAR.com).

EXAMPLE 2 Subcloning and Expression in B. Subtilis of the XyloglucanaseCore Part of the XYG1006 Multidomain Gene (Hereafter Called XYG1006)from Paenibacillus polymyxa Encoding for the Enzyme of the Invention

In order to express the xyloglucanase enzyme core part of the XYG1006multidomain enzyme the following PCR cloning scheme was followed. Thiscloning makes a translational fusion between the Amylase signal peptideon pPL3143 and the mature part of the XYG1006 multidomain enzyme. At thesame time it creates an artificial translational stop codoncorresponding to the amino acid number 560 in the XYG1006 multidomaingene.

The XYG1006 encoding DNA sequence was PCR amplified using the PCR primerset consisting of these two oligonucleotides:

XYG1006.upper.Pstl (SEQ ID NO: 15)5′-GCATTCTGCAGCAGCGGCTGTGGTTCACGGTCAAACGGC-3′ XYG1006.lower.Ascl (SEQ IDNO: 16) 5′-GCTAGGCGCGCCTACACTGGAGACGTGTCATTGCCAGTAG-3′

Restriction Sites PstI and AscI are Underlined.

pXYG1006 plasmid DNA from E. coli, DSM 13321, was used as template in aPCR reaction using Amplitaq DNA Polymerase (Perkin Elmer) according tomanufacturer's instructions. The PCR reaction was set up in PCR buffer(10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% (w/v) gelatine)containing 200 micro-M of each dNTP, 2.5 units of AmpliTaq polymerase(Perkin-Elmer, Cetus, USA) and 100 pmol of each primer.

The PCR reaction was performed using a DNA thermal cycler (Landgraf,Germany). One incubation at 94° C. for 1 min followed by thirty cyclesof PCR performed using a cycle profile of denaturation at 94° C. for 30sec, annealing at 60° C. for 1 min, and extension at 72° C. for 2 min.Five microliter aliquots of the amplification product was analysed byelectrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance of aDNA fragment size 1.6 kb indicated proper amplification of the genesegment.

Subcloning of PCR Fragment:

Forty-five microliter aliquots of the PCR products generated asdescribed above were purified using QIAquick PCR purification kit(Qiagen, USA) according to the manufacturer's instructions. The purifiedDNA was eluted in 50 microliters of 10 mM Tris-HCl, pH 8.5.

Five micrograms of pPL3143 and twenty-five microliters of the purifiedPCR fragment was digested with PstI and AscI, electrophoresed in 0.8%low gelling temperature agarose (SeaPlaque GTG, FMC) gels, the relevantfragments were excised from the gels, and purified using QIAquick Gelextraction Kit (Qiagen, USA) according to the manufacturer'sinstructions. The isolated PCR DNA fragment was then ligated to thePstI-AscI digested and purified pPL3143. The ligation was performedovernight at 16° C. using 0.5 microgram of each DNA fragment, 1 U of T4DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform competent B. subtilis PL1801cells. The transformed cells were plated onto LBPG agar platescontaining 10 micrograms/ml of Kanamycin and 0.2% AZCL-Xyloglucan(Megazyme). After 18 hours incubation at 37° C. colonies were seen onplates. Several clones showing a blue halo around the colony wereanalyzed by isolating plasmid DNA from overnight culture broth.

One such positive clone was re-streaked several times on agar plates asused above, this clone was called PL3344. The clone PL3344 was grownovernight in TY-10 microgram/ml Kanamycin at 37° C. The following day, 1ml of cells was used to isolate plasmid from the cells using the QiaprepSpin Plasmid Miniprep Kit #27106 according to the manufacturersrecommendations for B. subtilis plasmid preparations. This DNA was DNAsequenced and revealed the DNA sequence corresponding to the mature partof the Paenibacillus polymyxa xyloglucanase encoding domain correctlyinserted into the pPL3143 cloning vector (Domain 1-1680 of SEQ ID NO: 1.The Bacillus subtilis strain PL3344 is thus containing a plasmidenabling the expression of the xyloglucanase domain of the XYG1006 multidomain gene. The translation product is thus from amino acid 1 to 559(SEQ ID NO: 2) which after cleavage of the signal peptide is expected tobe secreted from B. subtilis as a protein spanning from amino acid 36 to559 (SEQ ID NO: 2).

PL3344 was grown in 25×200 ml BPX media with 10 micrograms/ml ofKanamycin in two 500 ml baffled shake flasks for 5 days at 37° C. at 300rpm.

EXAMPLE 3

Purification and Characterization of Xyloglucanase from PaenibacillusPolymyxa

The clone XYG1006 obtained as described in example 2 (PL3344 expressedin B. subtilis) was incubated in 4500 ml of PS-1 containing mg/mlkanamycin from shake flasks with a final pH of 5.5.

The fermentation medium was flocculated using cationic flocculationagent C521 (10% solution) and 0.1% solution of anionic agent A130: To4500 ml of broth, 200 ml of C521 was added (10%) simultaneously with 400ml of A130 (0.1%) under constant stirring at room temperature. Theflocculated material was separated by centrifugation using a Sorval RC3B centrifuge at 4,500 rpm for 30 minutes. The supernatant was adjustedto pH 7.0 using sodium hydroxide and then batch treated with 300 g ofHPQ Sepharose equilibrated with 50 mM tris pH 7.0, and the unboundmaterial was filter sterilized through a 0.7 micro-m filter.

The liquid was concentrated into 550 ml using filtron ultrafiltrationwith a MW cut off of 10 kDa. The concentrate was then diluted to 900 mland passed over an S-Sepharose column equilibrated with 25 mM sodiumacetate pH 5.0, and the not bound material was concentrated to 390 ml.

For obtaining a pure enzyme 2 ml of this partial pure enzyme was appliedto a size chromatography (Superdex 75) column equilibrated with 0.1 MSodium acetate pH 6.0. The xyloglucanase eluted as a single peak with aMW of 58 kDa in SDS-PAGE and with a specific activity of 255 XyloU unitsper mg protein.

The cloned xyloglucanase of the invention was used for raising rabbitantiserum.

After electroblotting of this band the N-terminal was determined asTAKTITIKVDTFKDRK (amino acids 41-56 of SEQ ID NO: 2)

This is in agreement with the amino acid sequence shown in SEQ ID NO: 2deduced from the DNA sequence shown in SEQ ID NO: 1 with a 40 amino acidpro sequence. The calculated MW from the deduced sequence was 58 kDa andthe calculated pl was 5.7. The molar extinction coefficient at 280 nmwas 105,640.

The enzyme melted in the DSC (Differential Scanning Calorimeter) at61.8° C. at pH 6.0.

The xyloglucanase showed optimal activity at 50° C. at pH 7.5 using thexylounits (XyloU) assay at different temperatures.

The xyloglucanase was more than 30% active between pH 5.0 and 8.0.

Kinetic determinations were performed using soluble tamarind gumxyloglucan at pH 7.5 and at 40° C., 20 minutes incubation time andmeasurement of the formation of reducing ends. The kCat of 220 per secwas determined. The kM was 0.2 g/l. The Michaelis-Menten kinetic valuescould be determined.

EXAMPLE 4 Stability and Activity of Paenibacillus polymyxa Xyloglucanasein Commercial Liquid Detergents

The xyloglucanase characterized in Example 3 was fully stable between pH5 and 10 at room temperature, and had a half life of more than 50 dayswhen incubated in a full formulated US liquid detergent as US Tide at30° C. The xyloglucanase was fully active in the commercial US liquiddetergent branded liquid Tide and in the commercial European liquiddetergent branded liquid Ariel, using 20 min. incubation at 40° C.

Literature

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1. An isolated xyloglucanase, which is any of (a) a polypeptide havingan amino acid sequence that is at least 80% identical with one or moreof the sequences of amino acids 40-559 of SEQ ID NO: 2, 4 or 6; and (b)a polypeptide encoded by a DNA sequence that hybridizes to one or moreof nucleotides 121-1677 of SEQ ID NO: 1, 3 or 5, under medium stringencyconditions, wherein the medium stringency conditions are defined ashybridization in 5×SSC at 45° C. and washing in 2×SSC at 60° C.
 2. Thexyloglucanase of claim 1, which has an amino acid sequence that is atleast 85% identical with amino acids 40-559 of SEQ ID NO:
 2. 3. Thexyloglucanase of claim 2, which has an amino acid sequence that is atleast 90% identical with amino acids 40-559 of SEQ ID NO:
 2. 4. Thexyloglucanase of claim 1, which has an amino acid sequence that is atleast 85% identical with amino acids 40-559 of SEQ ID NO:
 4. 5. Thexyloglucanase of claim 4, which has an amino acid sequence that is atleast 90% identical with amino acids 40-559 of SEQ ID NO:
 4. 6. Thexyloglucanase of claim 1, which has an amino acid sequence that is atleast 85% identical with amino acids 40-559 of SEQ ID NO:
 6. 7. Thexyloglucanase of claim 6, which has an amino acid sequence that is atleast 90% identical with amino acids 40-559 of SEQ ID NO:
 6. 8. Thexyloglucanase of claim 1, which is encoded by a DNA sequence thathybridizes to nucleotides 121-1677 of SEQ ID NO: 1 under mediumstringency conditions.
 9. The xyloglucanase of claim 1, which is encodedby a DNA sequence that hybridizes to nucleotides 121-1677 of SEQ ID NO:3 under medium stringency conditions.
 10. The xyloglucanase of claim 1,which is encoded by a DNA sequence that hybridizes to nucleotides121-1677 of SEQ ID NO: 5 under medium stringency conditions.
 11. Thexyloglucanase of claim 8, which is obtained from theBacillus/Lactobacillus subdivision.
 12. The xyloglucanase of claim 11,which is obtained from a species of Paenibacillus.
 13. The xyloglucanaseof claim 12, which is obtained from Paenibacillus polymyxa.
 14. Thexyloglucanase of claim 9, which is obtained from theBacillus/Lactobacillus subdivision.
 15. The xyloglucanase of claim 14,which is obtained from a species of Paenibacillus.
 16. The xyloglucanaseof claim 15, which is obtained from Paenibacillus polymyxa.
 17. Thexyloglucanase of claim 10, which is obtained from theBacillus/Lactobacillus subdivision.
 18. The xyloglucanase of claim 17,which is obtained from a species of Paenibacillus.
 19. The xyloglucanaseof claim 18, which is obtained from Paenibacillus polymyxa.
 20. Adetergent composition comprising a xyloglucanase of claim 1 and asurfactant.
 21. A process for washing a fabric, comprising treating thefabric during a washing cycle of a machine washing process with awashing solution which comprises a xyloglucanase of claim 1.